Targeted radiotherapy of neuroblastoma: future directions

No abstract available

The suppression of DNA repair induced by PARP-1 inhibitors rucaparib and olaparib in combination with the radiopharmaceutical 131I-MIBG in noradrenaline transporter-expressing xenograft tumors

No abstract available

Age-related mitochondrial DNA depletion and the impact on pancreatic beta cell function

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes

Inhibition of glycolysis and mitochondrial respiration promotes radiosensitisation of neuroblastoma and glioma cells

Background: Neuroblastoma accounts for 7% of paediatric malignancies but is responsible for 15% of all childhood cancer deaths. Despite rigorous treatment involving chemotherapy, surgery, radiotherapy and immunotherapy, the 5-year overall survival rate of high-risk disease remains < 40%, highlighting the need for improved therapy. Since neuroblastoma cells exhibit aberrant metabolism, we determined whether their sensitivity to radiotherapy could be enhanced by drugs affecting cancer cell metabolism. Methods: Using a panel of neuroblastoma and glioma cells, we determined the radiosensitising effects of inhibitors of glycolysis (2-DG) and mitochondrial function (metformin). Mechanisms underlying radiosensitisation were determined by metabolomic and bioenergetic profiling, flow cytometry and live cell imaging and by evaluating different treatment schedules. Results: The radiosensitising effects of 2-DG were greatly enhanced by combination with the antidiabetic biguanide, metformin. Metabolomic analysis and cellular bioenergetic profiling revealed this combination to elicit severe disruption of key glycolytic and mitochondrial metabolites, causing significant reductions in ATP generation and enhancing radiosensitivity. Combination treatment induced G2/M arrest that persisted for at least 24 h post-irradiation, promoting apoptotic cell death in a large proportion of cells. Conclusion: Our findings demonstrate that the radiosensitising effect of 2-DG was significantly enhanced by its combination with metformin. This clearly demonstrates that dual metabolic targeting has potential to improve clinical outcomes in children with high-risk neuroblastoma by overcoming radioresistance

An evaluation in vitro of PARP-1 inhibitors, rucaparib and olaparib, as radiosensitisers for the treatment of neuroblastoma

Background: The radiopharmaceutical 131I-meta-iodobenzylguanidine (131I-MIBG) is an effective treatment for neuroblastoma. However, maximal therapeutic benefit from 131I-MIBG is likely to be obtained by its combination with chemotherapy. We previously reported enhanced antitumour efficacy of 131I-MIBG by inhibition of the poly(ADP-ribose) polymerase-1 (PARP-1) DNA repair pathway using the phenanthridinone derivative PJ34. Recently developed alternative PARP-1 inhibitors have greater target specificity and are expected to be associated with reduced toxicity to normal tissue. Therefore, our purpose was to determine whether the more specific PARP-1 inhibitors rucaparib and olaparib enhanced the efficacy of X-radiation or 131I-MIBG. Methods: Radiosensitisation of SK-N-BE(2c) neuroblastoma cells or noradrenaline transporter gene-transfected glioma cells (UVW/NAT) was investigated using clonogenic assay. Propidium iodide staining and flow cytometry was used to analyse cell cycle progression. DNA damage was quantified by the phosphorylation of H2AX (γH2AX). Results: By combining PARP-1 inhibition with radiation treatment, it was possible to reduce the X-radiation dose or 131I-MIBG activity concentration required to achieve 50 % cell kill by approximately 50 %. Rucaparib and olaparib were equally effective inhibitors of PARP-1 activity. X-radiation-induced DNA damage was significantly increased 2 h after irradiation by combination with PARP-1 inhibitors (10-fold greater DNA damage compared to untreated controls; p < 0.01). Moreover, combination treatment (i) prevented the restitution of DNA, exemplified by the persistence of 3-fold greater DNA damage after 24 h, compared to untreated controls (p < 0.01) and (ii) induced greater G2/M arrest (p < 0.05) than either single agent alone. Conclusion: Rucaparib and olaparib sensitise cancer cells to X-radiation or 131I-MIBG treatment. It is likely that the mechanism of radiosensitisation entails the accumulation of unrepaired radiation-induced DNA damage. Our findings suggest that the administration of PARP-1 inhibitors and 131I-MIBG to high risk neuroblastoma patients may be beneficial

The effect of glibenclamide on insulin secretion following <i>TFAM</i> silencing-induced mtDNA depletion.

<p>Seventy two hours post transfection cells were stimulated with 3 mM or 25 mM glucose, supplemented with or without 0.1 µM glibenclamide. Insulin secretion was determined by insulin ELISA and normalised to whole cell protein content. Data shown are from 4 separated experiments performed in triplicate, and are normalised to Scrambled negative control cells stimulated with 3 mM glucose without glibenclamide. Data presented are means ± SEM. * p&lt;0.05, ** p&lt;0.01.</p

TFAM mRNA silencing induces mtDNA depletion 72 h post transfection.

<p>MIN6 cells were transfected with TFAM-193, TFAM-429 or Scrambled siRNA probes, or with no siRNA (Shocked). TFAM mRNA expression was quantified relative to reference gene <i>B2M</i> by real-time PCR at 48 h (A) and 72 h (B) post transfection. mtDNA depletion was also measured by real-time PCR, using mitochondrial encoded <i>ND5</i> relative to nuclear encoded <i>GAPDH</i> at 48 h (C) and 72 h (D) post transfection. All results normalised to Scrambled negative control. Experiment repeated once (C), twice (A) or 4 times (B, D) in triplicate. Data presented are means ± SEM (SD in (C)). * p&lt;0.05, *** p&lt;0.001.B2M, β2 Microglobulin; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; ND5, NADH Dehydrogenase 5; TFAM, Mitochondrial Transcription Factor A.</p

The effect of partial mtDNA depletion on glucose-stimulated insulin secretion.

<p>Seventy two hours post transfection cells were stimulated with basal (3 mM) or high (25 mM) glucose concentrations. Insulin secretion (A) and insulin content (B) were determined by insulin ELISA and normalised to protein content. Data normalised to 3 mM glucose stimulated Scrambled control cells. mtDNA levels (C) and <i>Ins1</i> insulin gene expression (D) were quantified and normalised to the Scrambled control. Data shown are from 9 (A) or 3 (B, C, D) separate experiments, each performed in triplicate. Data presented are means ± SEM. ** p≤0.01, *** p&lt;0.001.</p

The effect of partial mtDNA depletion on mitochondrial gene transcription and protein translation.

<p>Mitochondrial DNA was depleted following <i>TFAM</i> silencing and COX1 mRNA expression was quantified relative to reference gene <i>B2M</i> 72 h post transfection (A). Protein was extracted 72 h post transfection and analysed by western blotting, probing for COX1, SDH70, and β-Actin proteins. Protein bands were quantified by densitometry, and optical density readings used to calculate the ratio of COX1 (B) and SDH70 (C) mitochondrial proteins relative to β-Actin loading control. A representative blot is shown in (D). Data in (A) are normalised to Scrambled control cells. Both experiments repeated 3 times, with each experimental repeat performed in triplicate (A) or duplicate (B, C). Data presented are means ± SEM. * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001. COX1, Cytochrome <i>c</i> Oxidase 1; SDH70, Succinate Dehydrogenase 70 kDa subunit.</p

The effect of partial mtDNA depletion on mitochondrial function.

<p>Cells were harvested 72 h post transfection and oxygen consumption rate (OCR) was measured using the Seahorse XF24 Analyzer. OCR in TFAM-429 cells (n = 8) was severely impaired compared to that of Scrambled (n = 8) and Shocked (n = 8) control cells (A). Mitochondrial activity was measured following injection of oligomycin, an inhibitor of Complex V (ATP Synthase), followed by two sequential injections of FCCP to uncouple respiration and induce maximal respiration, and finally antimycin, an inhibitor of Complex III (Ubiquinol-Cytochrome <i>c</i> Reductase) preventing electron transfer and subsequently abolishing the proton gradient required for ATP synthesis. Basal and maximal respiratory capacity (B) and ATP synthesis by oxidative phosphorylation (OXPHOS) (C) were calculated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115433#pone.0115433-Bonnen1" target="_blank">[30]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115433#pone.0115433-Brand2" target="_blank">[32]</a>. Data were normalised to protein concentration and are presented means ± SEM. * p&lt;0.5, ** p&lt;0.001, *** p&lt;0.0001.</p